(1) Field of the Invention
The present invention relates to a process for the isolation and preferably purification of taxol and other taxanes from Taxus spp plant material by using a particular combination of solvent extraction and preferably normal phase chromatographic purification which produces the taxanes in high yield and purity. The process is particularly characterized in the use of preliminary solvent extraction and purification steps which remove the desired taxanes from the plant material without the lipid and chlorophyll components which interfere with the chromatographic purification.
(2) Description of the Prior Art
The production of taxol from ornamental yew needles, barks and roots at present is not economical due to an extremely high percentage of unwanted impurities carried forward in the extract (40-50% by weight of the dried plant material) during the extraction. This unusually high percentage of impurities in the solvent extract of the needles of ornamental yew makes it very expensive and uneconomical to purify taxol and taxanes from this source in addition to the high cost in drying the needles. Published reports suggest that ornamental yew needles contain about 0,002 to 0.01% of taxol on a dry weight basis (Witherup, S., et al., journal of Natural Products 53, 1249-1255 (1990). Organic solvent extraction of 1 kg of the dried ornamental yew needles will afford about 450-500 g of the extract after removing the solvent (45-50% to the biomass) by the published extraction methods using 95% ethanol in water.
The prior art has described taxol and other taxanes isolated from the bark of Taxus spp. which are useful as chemotherapeutic agents, particularly in the treatment of cancers. Illustrative are U.S. Pat. No. 5,019,504 (1991) to Christen et al and WO 92/07842 (1992) to Rao et al. Christen et al describe a cell culture process using Taxus brevifolia for producing the taxanes which are then separated by chromatography, by solvent extraction or adsorption methods. Culturing of plant cells is a difficult method for production of the taxanes for use on a large scale. Rao et al describe a process using reverse phase liquid chromatography. The plant material is extracted with a polar solvent, which is 95% by volume ethanol, for 24 hours at ambient temperatures. The 95% by volume ethanol in this step removes many extraneous lipid components and chlorophyll. A solvent-solvent extraction or partitioning step is then used to remove water soluble materials from the water insoluble taxanes. Various solvents are described for the taxanes (chloroform, benzene, ligroin). The solvent is removed to produce a crude extract. This crude extract is then subjected to the reverse phase chromatography in a solvent mixture to isolate the individual taxanes.
There are multiple problems with the Rao et al process. The most important is that plant lipid components and large quantities of chlorophyll are extracted by 95% by volume ethanol. These lipid and chlorophyll components interfere with the separation in the chromatographic column. Also, the crude product is colored from compounds in the plant material and these color compounds interfere with the chromatographic separation. The plant material is preferably dried to less than 0.5% moisture and ground, which aids in the removal of the taxanes during the initial extraction. Drying the Taxus plant material is an expensive step. The extraction process of Rao produces large quantities of crude extract in which taxol and taxanes are only a minor component. The reverse phase chromatographic separation using the process of Rao et al is such that taxol is not cleanly separated. The mother liquor from the initial separation of the taxol is subjected to additional reverse phase chromatography and recrystallization to separate more taxol. The more polar solvent fractions contain 10-deacetylbaccatin III which can be crystallized to remove this compound and then resubjected to standard chromatography. Multiple reverse phrase liquid chromatographic steps may be necessary in the process of Rao et al.
In another disclosed method (FIG. 2) ligroin is used to remove lipid components. Aqueous methanol removes the crude taxane mixture which is then extracted with less polar solvents to separate taxol and related compounds from 10 deactylbaccatin III. The taxanes can be isolated and recrystallized using the reverse phase chromatography. An aqueous methanol extract is partitioned between water and benzene and then extracted with chloroform and the solvents are removed. Methanol or acetonitrile and water is used in reverse phase liquid chromatography to separate the taxanes.
The procedures are set forth in FIGS. 1 to 3 of this reference. None of the procedures provide a clean separation of each of the components in a single pass through a column without a solvent-solvent extraction. Large amounts of various solvents are necessary.
Reverse phase chromatographic separation of impure taxanes from plant materials is very expensive because of the cost of the column materials. Generally reverse phase separation can be used on the bark of Pacific Yew because of the relatively low concentration of pigments, lipids and waxes and high concentration of taxol; however, the yew needles contain lesser amounts of taxol and significant amounts of impurities and thus reverse phase chromatography for separation of taxol from the bulk of the yew materials other than Pacific Yew bark is not practical. There is an urgent need for lower cost production of taxol.
Reverse phase separation is economical only where relatively small numbers of compounds in a mixture are to be separated. A preferred material is silica particles coated with octadecyl silane which is expensive. These particles are used in a column usually at high pressures of between about 50 and 6000 psi and usually with a mixture of acetonitrile and water. In reverse phase chromatography the most polar compounds pass through the column the fastest in contrast to normal phase chromatography.
In normal phase chromatography very inexpensive silica gel is used which is about 100 times or more less expensive than the ordinary reverse phase particles. In normal phase, the silica gel contains silylhydroxide groups (--SiOH) which bind with polar groups of the solute. Thus the more polar compounds move more slowly along the column than less polar compounds. It would be highly desirable to be able to use normal phase chromatography for the separation of taxol and related compounds which are semi-polar; however, to date such columns have not been used because of the large number of polar impurities in the yew plant material, particularly in ornamental yew.
The problem is to simplify the procedures used to produce taxol and other taxane derivatives and reduce the cost using simplified extraction and chromatographic techniques.